Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2

Description

. Quality assessment of placeholder peptide-loaded HLA tetramers. Assessment by (A) analytical SEC and (B) SDS-PAGE analysis show a highly multimeric structures with little aggregation. (B) SDS-PAGE analysis of placeholder peptide-loaded HLA tetramers either boiled and reduced (B/R), or non-boiled and non-reduced (NB/NR), in which the streptavidin quaternary structure is maintained. Densitometry analysis indicates all preparations are >80% trimer/tetramer fraction.

Fig. S2. Assessment of UV-mediated peptide-exchange tetramers. (A)
Cell binding with peptideexchanged HLA tetramers on corresponding peptide-expanded, healthy donor T cells. Placeholder-loaded HLA tetramers ("No UV") were UV-exchanged as indicated in the Methods, either in the presence ("UV + PEP") or absence ("UV/No peptide") of rescue peptide, and used to stain the indicated peptide-expanded cells. Peptide abbreviations and sources: NLVPMVATV (NLV, CMV pp65), ELAGIGILTV (ELA, MART-1), YMLDLQPETT (YML, HPV16 E7), SLPITVYYA (SLP, HSV gD1), RMFPNAPYL (RMF, Wilms tumor antigen 1), VTEHDTLLY (CMV pp50), QYDPVAALF (CMV pp65), RPHERNGFTVL (CMV pp65). (B-D) Comparison of cell staining with "industry standard" biotin-mediated tetramers that were produced by refolding with a cognate peptide ("Cognate"), placeholder-loaded covalent tetramers ("Placeholder"), placeholder-to-cognate UV-exchanged covalent tetramers ("UV-Exchanged"), or secondary antibody only ("Secondary Only"). (B) Mean fluorescence intensity (MFI) of NLVexpanded cells stained with titrations of A*02:01 tetramers. (C) Comparison of MFI of peptideexpanded cells stained with corresponding A*01:01, A*24:02 and B*07:02 tetramers at 20nM. (D) Cytometry scatter plots corresponding to the A*24:02 data represented in panel C, comparing the staining of cells expanded with peptide QYDPVAALF and incubated with biotinmediated or covalent, UV-exchanged tetramers.     Putative hit identification. Z-scores for each cell-pMHC interaction are calculated across all cells and all tetramers and plotted on the x-axis and y-axis, respectively. A classifier score for each clonotype (possibly multiple cells) is calculated using a product of both Z-scores (each averaged across cells within clonotype) and the number of cells represented by the clonotype. Shown is a representative analysis for a convalescent subject sample stained with the B*07:02 tetramer library, including a global analysis across cells and several individual clonotypes isolated from the global plot for epitopes RPPIFIRRL and SPRWYFYYL (shown in red in respective plots).

Fig. S8. Assessment of frequency calculation for Influenza A specific CD8 + T cells in a wellcharacterized sample. (A)
Multiclonal T cells were characterized by direct encapsulation and single-cell sequencing to determine frequency of TCR clonotypes. In parallel, these cells were stained with an A*01:01 tetramer library containing two epitopes for Influenza A, tetramer positive cells were sorted, and reactivities were determined using the approach described in the Methods section. Frequencies of the parent sample were calculated by taking the product of the fraction of the reactive clonotype in the sorted sample and fraction of tetramer (+) cells sorted. (B) Table of putative hits quantified in sorted (calculated, %) and unsorted (measured, %) samples. Table S1. Putative hits identified by antigen, representing the number of clonotypes that were found to be specific to any epitope within the given antigen. Data file S1.
Raw data from manuscript figures.

Data file S2.
Subject summary and metadata.

Data file S3.
Putative TCR-peptide-HLA hits identified in this study.

Data file S4.
Summary of SARS-CoV-2 epitopes identified in this study compared to those obtained in other publications. The ranking of epitopes is a qualitative measure of relevance by HLA, calculated using the number of clonotypes (Francis), frequency of positive response (Tarke and Feretti), and p-value (Saini).

Data file S5.
Epitopes and hits discovered in this work that would be impacted by high-threat SARS-CoV-2 variants. Shown are the epitopes included in this study that align with the specified mutation along with the number of clonotypes found to bind that epitope across the specified number of cells.

Data file S6.
Recombinant TCR-peptide-HLA interactions confirmed by functional testing. Shown are the cohort, patient identifier, HLA, epitope, antigen, and alpha/beta CDR3 sequences for interactions originally identified across nCells as well as percentage of TCR transduced cells found to be CD69+ or expressing NFAT after stimulation. Thresholds for positive interactions were 20% positive and 3-fold over DMSO for CD69 expression and NFAT signaling, respectively.

Data file S7.
Representation of transcriptional phenotype by epitope specificity and cohort. These values are consistent with the cells/clusters represented in Figure 5 of the main text.

Data file S8.
Composition of tetramer libraries indicating HLA allele, peptide sequence, and DNA barcode.

Data file S9.
TCR sequences for anti-MART-1 T cells used in sample multiplexing. Sequences annotated indicate the top clonotypes (representing ~99%) of a cell line expanded using peptide ELAGIGILTV in A*02:01.